Structural and functional analyses of photosynthetic regulatory genes regA and regB from Rhodovulum sulfidophilum, Roseobacter denitrificans, and Rhodobacter capsulatus

Abstract

Genes coding for putative RegA, RegB, and SenC homologues were identified and characterized in the purple nonsulfur photosynthetic bacteria Rhodovulum sulfidophilum and Roseobacter denitrificans, species that demonstrate weak or no oxygen repression of photosystem synthesis. This additional sequence information was then used to perform a comparative analysis with previously sequenced RegA, RegB, and SenC homologues obtained from Rhodobacter capsulatus and Rhodobacter sphaeroides. These are photosynthetic bacteria that exhibit a high level of oxygen repression of photosystem synthesis controlled by the RegA-RegB two-component regulatory system. The response regulator, RegA, exhibits a remarkable 78.7 to 84.2% overall sequence identity, with total conservation within a putative helix-turn-helix DNA-binding motif. The RegB sensor kinase homologues also exhibit a high level of sequence conservation (55.9 to 61.5%) although these additional species give significantly different responses to oxygen. A Rhodovulum sulfidophilum mutant lacking regA or regB was constructed. These mutants produced smaller amounts of photopigments under aerobic and anaerobic conditions, indicating that the RegA-RegB regulon controls photosynthetic gene expression in this bacterium as it does as in Rhodobacter species. Rhodobacter capsulatus regA- or regB-deficient mutants recovered the synthesis of a photosynthetic apparatus that still retained regulation by oxygen tension when complemented with reg genes from Rhodovulum sulfidophilum and Roseobacter denitrificans. These results suggest that differential expression of photosynthetic genes in response to aerobic and anaerobic growth conditions is not the result of altered redox sensing by the sensor kinase protein, RegB.

FIG. 1

Physical and genetic maps of the photosynthetic regulatory gene cluster. ORFs and their directions of transcription are represented by open arrows. The broken lines shown in Rhodovulum (Rdv.) sulfidophilum and Roseobacter (Rsb.) denitrificans indicate the unsequenced regions. The DNA template used in the Southern hybridization is indicated by a thick line shown in Rhodobacter (Rba.) capsulatus. Rhodobacter (Rba.) sphaeroides prrB, prrC, prrA, and spb genes are thought to be equivalent to the homologous regB, senC, regA, and hvrA genes, respectively (see the text).

FIG. 2

Alignment of amino acid sequences of RegA of Rhodobacter capsulatus (Rba. cap) (55), Rhodovulum sulfidophilum (Rdv. sul), and Roseobacter denitrificans (Rsb. den), PrrA of Rhodobacter sphaeroides (Rba. sph) (19), ActR of Rhizobium meliloti (R. mel) (65), and RegR of Bradyrhizobium japonicum (B. jap) (5). Asterisks indicate identical amino acids. Residues considered functional and a predicted helix-turn-helix DNA-binding motif are boxed.

FIG. 3

Alignment of amino acid sequences of RegB of Rhodobacter capsulatus (Rba. cap) (42), Rhodovulum sulfidophilum (Rdv. sul), and Roseobacter denitrificans (Rsb. den), PrrB of Rhodobacter sphaeroides (Rba. sph) (20), ActS of Rhizobium meliloti (R. mel) (65), and RegS of Bradyrhizobium japonicum (B. jap) (5). Asterisks indicate identical amino acids. The histidine block (H), an asparagine-rich block (N), glycine-rich domains (G1 and G2), and a variable-length spacer (F) which are roughly conserved in the histidine kinases (45, 46) are boxed.

FIG. 4

Alignment of amino acid sequences of SenC of Rhodobacter capsulatus (Rba. cap) (9), Rhodovulum sulfidophilum (Rdv. sul), and Roseobacter denitrificans (Rsb. den), PrrC of Rhodobacter sphaeroides (Rba. sph) (20), and yeast nucleus-encoded protein SCO1 (8, 54). Asterisks indicate identical amino acids. The residues hypothesized to contribute to a predicted iron-binding domain are boxed (9).

FIG. 5

Growth profiles of Rhodovulum sulfidophilum wild-type, regA-disrupted (RESA1), and regB-disrupted (RESB20) strains. Cells were grown under anaerobic high-intensity light conditions (100 W/m²) (solid circles, wild-type cells; solid squares, RESA1 cells; solid triangles, RESB20 cells) (A) and anaerobic low-intensity light conditions (3 W/m²) (open circles, wild-type cells; open squares, RESA1 cells; open triangles, RESB20) (B).

FIG. 6

Absorption spectra of Rhodovulum sulfidophilum wild type (solid lines), RESA1 (short dashed lines) and RESB20 (long dashed lines) grown under anaerobic low-intensity light conditions (3 W/m²) (A), anaerobic high-intensity light conditions (100 W/m²) (B), aerobic-dark conditions (C), and aerobic high-intensity light conditions (100 W/m²) (D). Cells in the mid-logarithmic growth phase were harvested and sonicated, and spectra were measured in membrane preparations. All samples contained 75 μg of protein per ml.